CN116790747B - Clca2作为靶标在治疗早发性卵巢功能不全中的应用 - Google Patents
Clca2作为靶标在治疗早发性卵巢功能不全中的应用 Download PDFInfo
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Abstract
本发明属于生物医药领域，具体涉及CLCA2作为靶标在治疗早发性卵巢功能不全中的应用。具体地，本发明提供了CLCA2作为靶标在治疗早发性卵巢功能不全中的应用，以及TP63作为靶标在诊断早发性卵巢功能不全中的应用。CLCA2是介导TP63突变导致细胞凋亡的重要分子，抑制CLCA2可以有效减少细胞凋亡，进而减少POI的发生。
Description
技术领域
本发明属于生物医药领域，具体涉及CLCA2作为靶标在治疗早发性卵巢功能不全中的应用。
背景技术
早发性卵巢功能不全(POI)是指女性在40岁之前发生卵巢功能的衰退和停滞，表现为至少4个月以上的闭经，卵泡刺激素(FSH)水平在大于4周的两次检测中均高于25IU/L，且伴有雌激素水平降低。POI发病率为1-5％，已经成为当代女性不孕的一个重要原因。POI的致病因素包括遗传因素、辐射、化疗、免疫、感染、卵巢手术因素等。近10-20年，随着全外显子组测序技术的发展，研究者们发现了几十个POI致病基因，并对部分基因进行了致病机制的研究。这些研究加深人们对POI疾病发病机理的认识，并推动了POI疾病治疗靶点和通路的选择。但POI的治疗方面却进展缓慢，目前尚无有效的治疗手段，患者的病情一旦进入至卵巢早衰阶段，其生育的可能性会变得非常渺茫。
TP63是p53转录因子家族的一员，可调控卵母细胞的生物学功能。TP63蛋白包含N末端转录激活结构域(TAD)和C末端的不育α基序结构域(SAM)，以及转录抑制结构域(TID)。TP63有多种变体(isoform)，其中TP63α是TP63的最长且功能最重要的变体，具有包含SAM和TID结构域的C末端区域。TP63也受到严格的调控，通常以二聚体的形式维持在非活性状态。当卵母细胞经历DNA损伤时，TP63可以诱导该卵母细胞发生凋亡。以往研究表明TP63蛋白C末端的截短突变可导致POI，包括R594*、Q568fs*3、W598*、R643*等。近期的一项研究发现TP63的某些错义突变(R97P、R647C)会产生类似截短突变的功能，使TP63的转录激活能力增强，导致POI。小鼠Tp63蛋白的C末端缺失导致Tp63以四聚体的形式持续性激活，加速卵母细胞凋亡。但TP63截短突变如何导致生殖细胞过快凋亡的具体机制尚不清晰。
发明内容
本发明的目的在于克服现有技术缺陷，提供CLCA2作为靶标在治疗早发性卵巢功能不全中的应用、TP63作为靶标在诊断早发性卵巢功能不全中的应用。具体地，本发明的技术方案如下：
一方面，本发明提供了检测CLCA2表达量的试剂在制备诊断早发性卵巢功能不全产品的应用。
优选地，本发明所述检测表达量的方法包括检测mRNA表达量的方法和/或检测蛋白表达量的方法。
优选地，所述mRNA表达量的检测方法包括：基于PCR的检测方法、Southern杂交方法、Northern杂交方法、点杂交方法、荧光原位杂交方法、DNA微阵列方法、ASO法、高通量测序平台方法。
优选地，所述检测蛋白表达量的方法包括：ELISA检测、Elispot检测、Western印迹或表面等离子共振法。
优选地，所述早发性卵巢功能不全是TP63突变型的早发性卵巢功能不全，所述TP63突变型的早发性卵巢功能不全患者具有TP63突变或表达TP63突变体，所述TP63突变是c.1742_1746+9del，所述TP63突变的序列如SEQ ID NO.3所示。
具体地，在患有所述TP63突变型的早发性卵巢功能不全的患者的样本中，所述CLCA2的表达量升高。
优选地，所述诊断是通过收集受试者样本进行检测而进行的。
优选地，所述样品包括：外周血、组织、血液、血清、血浆、尿液、唾液、精液、乳汁、脑脊髓液、泪液、痰、粘液、淋巴、胞液、腹水、胸膜积液、羊水、膀胱冲洗液和支气管肺泡灌洗液。
另一方面，本发明提供了CLCA2的抑制剂在制备治疗早发性卵巢功能不全(POI)的产品中的应用。
优选地，所述治疗产品是药物或药物组合物，所述药物组合物中还可以包含其他活性物质从而达到提升治疗效果的作用。
具体地，可通过任何能够使CLCA2抑制剂到达靶细胞的方法将CLCA2的抑制剂施用给患者。这些方法包括但不限于经口、直肠、经鼻、外敷、皮内、皮下、静脉内、肌内、气管内和腹腔内施用方式。所述抑制剂可以溶于诸如氯化钠、林格氏溶液、葡萄糖、葡萄糖和氯化钠、水、盐水、林格氏(Ringer’s)溶液、葡萄糖溶液的水性媒剂进行施用，也可溶于脂质体和诸如固定油、棉籽油、芝麻油或花生油和酯类等非水性媒剂进行施用。另外，还可添加各种能够增强组合物的稳定性、无菌性和等渗性的添加剂，包括抗菌防腐剂、抗氧化剂、螯合剂以及缓冲剂。然而，所用的任何媒剂、稀释剂或添加剂皆必须具有生物相容性并可与本发明的抑制剂相容。
本发明所述CLCA2抑制剂可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。所述CLCA2抑制剂的给药剂量根据给药对象、给药途径或药物的制剂形式不同进行变化，但以保证该药物组合物在哺乳动物体内能够达到有效的血药浓度为前提。所述药物组合物可以是任何剂型，并以任何方式施用。
另一方面，本发明提供了抑制细胞凋亡的方法，所述方法包括将CLCA2抑制剂与目标细胞接触。
优选地，所述方法是体外进行的。
优选地，所述方法是非治疗目的的。
优选地，所述细胞凋亡的程度通过任意常规的检测手段而确定，本发明具体实施例中通过Annexin V测定而确定细胞凋亡的程度。
优选地，所述接触是指通过转染(转导)的方式使CLCA2抑制剂进入细胞。
优选地，所述目标细胞是动物细胞。
优选地，所述动物包括人、小鼠、大鼠、豚鼠、兔、猿、猴、黑猩猩、牛、羊、猪、马、犬、猫、羊驼等。
优选地，所述目标细胞是人类细胞系。
优选地，所述人类细胞系包括293细胞、293T细胞、293FT细胞、293LTV细胞、293EBNA细胞及其他的从293细胞分离的克隆；SW480细胞、u87MG细胞、HOS细胞、C8166细胞、MT-4细胞、Molt-4细胞、HeLa细胞、HT1080细胞、TE671细胞。
优选地，所述目标细胞是具有TP63突变体的细胞，或者，表达TP63突变体(TP63-Mut)的细胞。
根据本发明，术语“抑制剂”指生物学的或化学的试剂，其能抑制或减少CLCA2的活性或者抑制CLCA2的表达。具体可以通过诸如RT-PCR或蛋白印迹分析的常规表达量检测方法测量CLCA2的表达。
具体地，本发明所述抑制剂包括特异性靶向CLCA2的多肽、多核苷酸或小分子；例如，结合CLCA2的抗体、反义寡核苷酸、低分子量分子(LMW)、siRNA、适配体、小分子化合物等。
更优选地，本发明通过如SEQ ID NO.1或SEQ ID NO.2所示的CLCA2siRNA序列特异性靶向CLCA2，降低CLCA2的表达量。
本文中使用的术语“治疗”通常涉及治疗人类或动物(例如，被兽医所应用)，其中前述CLCA2可达到某些预期的治疗效果，例如，抑制病症的发展(包括降低发展速度、使发展停止)、改善病症和治愈病症。还包括作为预防措施(例如预防)的治疗。对还没有发展为病症但有发展为该病症危险的高风险人群的用途，也包括在术语“治疗”中。
另一方面，本发明提供了TP63截断突变或TP63突变体的检测试剂在制备诊断早发性卵巢功能不全产品的应用，所述TP63突变的序列如SEQ ID NO.3所示，所述TP63截短突变是c.1742_1746+9del。
更具体地，具有所述TP63截短突变的TP63突变体(TP63-Mut)序列如SEQ ID NO.3所示。则具有所述TP63截短突变的受试者诊断为POI患者或具有较高的POI风险
在某些非限制性实施方案方式中，所述的产品包括试剂盒、芯片、试纸。在某些实施方式中，所述试剂盒包括qPCR试剂盒。
优选地，所述诊断是通过收集受试者样本进行检测而进行的。
优选地，所述样品包括：外周血、组织、血液、血清、血浆、尿液、唾液、精液、乳汁、脑脊髓液、泪液、痰、粘液、淋巴、胞液、腹水、胸膜积液、羊水、膀胱冲洗液和支气管肺泡灌洗液。
优选地，所述检测试剂中包括引物。更具体地，特异性扩增TP63的引物，所述“特异性扩增”包括通过引物仅扩增目的基因，而不扩增其他基因。
更优选地，所述检测试剂还包括PCR扩增所需要的试剂。优选地，所述PCR扩增所需要的试剂优选包括但不限于dNTP，PCR缓冲液，镁离子和Tap聚合酶。本发明在具体实施过程中，可根据实际需要，常规选择其他试剂。
本发明对所述扩增的方法没有特殊限定，采用本领域中常规扩增方法即可，示例性的扩增方法如PCR，更具体地包括反转录PCR(RT-PCR)，原位PCR，连接酶链反应(Ligasechain reaction，LCR)，标记PCR(Labeled primers，LP-PCR)，反向PCR(reverse PCR，扩增两引物外侧未知序列)，不对称PCR(asymmetric PCR)，降落PCR(touchdown PCR)，重组PCR(recombinant PCR)，巢式PCR(nest PCR)，多重PCR(multiplex PCR)，免疫-PCR(immuno-PCR)，mRNA差异PCR，链替换扩增(Strand displacement amplification，SDA)，依赖核酸序列的扩增(Nucleic acid sequence-based amplification，NASBA)，转录依赖的扩增系统(Transcript-based amplification system，TAS)，Q复制酶(Q-beta replicase)催化RNA扩增，滚环扩增(Rolling circle amplification，RCA)，环介导的等温扩增(Loopmediated isothermal amplification，LAMP)等。
本发明所述“诊断”包括疾病风险的预测、疾病发病与否的诊断、还包括对疾病预后的评估。
本发明所述“截短突变”也可称为“突变”，具体是指野生型的多核苷酸序列发生改变，成为变异体，变异体可以是天然发生的或非天然发生的，具有本发明所述TP63截短突变的TP63突变体(TP63-Mut)序列如SEQ ID NO.3所示。
本发明所述“早发性卵巢功能不全(primary ovarian insufficiency，POI)”也可称为原发性卵巢功能不全，是指女性在40岁前，由于卵巢功能减退引起的一系列临床综合征，具体表现在显性POI指发生月经不规律、血清促性腺激素水平升高及生育力降低。更优选地，患有本发明所述早发性卵巢功能不全的患者具有TP63突变或表达TP63突变体，所述TP63突变是c.1742_1746+9del，所述TP63突变的序列如SEQ ID NO.3所示，在具体实施例中可以称为p.S551Rfs*6。在本发明中可以将以上特定类型的早发性卵巢功能不全称为TP63突变型的早发性卵巢功能不全。
本发明的有益效果是：
1、本发明中，CLCA2是介导TP63突变导致细胞凋亡的重要分子，使用靶向CLCA2的siRNA敲低CLCA2基因的表达水平，可以有效减少细胞凋亡，进而减少POI的发生。CLCA2作为治疗靶标在制备治疗POI的试剂盒中的应用前景广阔。
2、本发明的TP63是POI的分子诊断的有效基因，扩展了研究人员对卵泡发生中细胞凋亡的了解，将直接有益于临床医生的精确诊断。
附图说明
图1是对POI患者携带TP63基因14bp的缺失突变的测序结果图。
图2是minigene的结构示意图。
图3是缺失突变导致TP63基因第13号外显子剪接过程异常的检测结果图。
图4是对剪接进行Sanger测序的测序结果图。
图5是TP63野生型蛋白和截短蛋白在细胞中的表达情况检测结果图。
图6是TP63截短突变导致细胞凋亡水平显著升高图
图7是Annexin V阳性细胞的免疫染色检测结果图。
图8是TP63野生型表达细胞和截短突变表达细胞间差异基因的分析结果图。其中，A-B：通过RNA-seq分析发现，与TP63野生型表达细胞相比，TP63截短突变表达细胞显著上调265个基因表达，下调139个基因表达；C：热图显示TP63野生型表达组(TP63-WT)和TP63截短突变表达组(TP63-Mut)的差异表达基因情况；D：GO分析差异表达基因富集的生物学过程和信号通路。
图9是CLCA2在TP63-WT和TP63-Mut细胞中的的表达情况检测结果图。
图10是通过siCLCA2抑制CLCA2表达后，细胞凋亡情况的检测结果图。
图11是ATM抑制剂抑制CLCA2表达后，细胞凋亡情况的检测结果图。
图12是本发明所中提出的TP63截短蛋白激活CLCA2进而引起细胞凋亡的分子机制图。A：TP63截短蛋白因缺少C-末端自抑制区，形成可组成型激活的四聚体，激活更多下游基因的表达，并导致细胞凋亡。CLCA2是被显著激活的下游基因；B：敲低CLCA2或使用ATM抑制剂可显著的降低因高表达TP63截短蛋白引起的细胞凋亡水平，所以CLCA2是介导TP63截短突变导致细胞凋亡的关键因子。
具体实施方式
下面结合实施例对本发明做进一步的说明，以下所述，仅是对本发明的较佳实施例而已，并非对本发明做其他形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容，依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化，均落在本发明的保护范围内。
本发明通用实验方法
1、细胞培养
293FT细胞在补充有10％胎牛血清、GlutaMax、MEM NEAA和Pen/Strep双抗的DMEM培养液中于5％二氧化碳条件下培养。
2、质粒转染和siRNA转染
293FT细胞接种在每孔2.5×105个细胞的六孔板中。转染时细胞密度为20-40％。使用jetPRIME(101000046；Polyplus，Illkirch，France)进行转染，按照制造商的说明书进行。将两微克DNA稀释到200μl jetPrime缓冲液中，并通过涡旋混合。接下来，加入4μljetPrime，涡旋10s，短暂旋转，并将混合物在22℃下孵育10分钟。然后，向每个孔中加入200μl转染混合物并均匀分布。轻轻搅拌平板，如果需要，4小时后用细胞生长培养基替换转染培养基，然后返回培养箱。48小时后收集细胞用于逆转录定量聚合酶链反应(RT-qPCR)和蛋白质免疫印迹。
3、荧光定量PCR
使用HiPure Total RNA Mini试剂盒(R4111-03；Magen，中国)提取总RNA，并根据制造商的方案使用TransScript One Step gDNA Removation and cDNA SynthesisSuperMix(AT311-03；全式金，中国北京)合成cDNA。使用PerfectStart Green qPCR SuperMix(AQ601-04；全式金，中国北京)在荧光定量-qPCR仪器(LightCycler 480II；罗氏，瑞士巴塞尔)上进行RT-qPCR反应。以ACTB基因作为管家基因。TP63的引物序列是：F-GGACCAGCAGATTCAGAACGG，R-AGGACACGTCGAAACTGTGC；CLCA2的引物序列是：F-GTGCATGGGATGTAATCACAGA，R-CAGCACTAAACAGACCACTTTGT；ACTB的引物序列是：F-GCACAGAGCCTCGCCTT，R-GTTGTCGACGACGAGCG。
4、免疫印迹分析
用质粒转染293FT细胞48小时。在含有cOmplete蛋白酶抑制剂鸡尾酒片(04693124001；Roche，德国)的RIPA缓冲液(R0020；北京索莱宝科技有限公司，中国)中制备总细胞裂解物。使用BCA测定法对蛋白质浓度进行定量(P0009；碧云天，中国)。等量的裂解物在10–12％的SDS-PAGE上电泳，并转移到0.2μm聚偏氟乙烯(PVDF)膜上(ISEQ00010；默克Millipore Ltd，Tullagreen，Cork Ireland)。用1:1000稀释液的适当一级抗体在4℃下孵育过夜。使用抗DYKDDDDK-Tag(9A3)小鼠mAb的抗体(#8146；Cell Signaling Technology，美国)和CLCA2多克隆抗体(19273-1-AP；ProteinTech，中国武汉)。然后用1×TBST洗涤膜，然后用抗小鼠IgG(h+L)生物素化抗体(ZB-2305；中杉金桥生物技术有限公司，中国)或抗兔IgG(h+R)生物素性抗体(ZB-2301；中杉金桥生物技术有限公司，中国)加入第二抗体1小时。使用Immobilon Western HRP底物试剂(WBKLS0500；Millipore，Billerica，美国)和ChemiDoc成像系统(Bio-Rad，Hercules，USA)对蛋白质进行可视化。
5、细胞凋亡实验
根据试剂盒制造商的方案，使用Annexin V-EGFP细胞凋亡检测试剂盒(C1067M；碧云天，中国)测定细胞凋亡。将细胞以2.5×105个细胞/孔的密度接种在六孔板中，然后用载体或质粒处理48小时。收获细胞并用100μl Annexin V在黑暗中染色15分钟。使用FlowJo_v10.8.1软件(BD，Ashland，美国)分析Annexin V的表达。
6、RNA测序和数据分析
委托北京贝瑞和康生物技术有限公司进行RNA测序和数据分析。提取总RNA，并使用Agilent 2100生物分析仪(Agilent Technologies，美国)分析RNA质量。使用IlluminaNovaSeq 6000测序平台构建cDNA文库。每组设3个生物学重复。使用具有Oligo(dT)的磁珠进行真核mRNA富集；将信使核糖核酸分解成短片段，以分割的信使核糖核酸为模板合成第一条cDNA链，然后加入缓冲溶液、dNTP和酶合成第二条cDNA链。纯化得到的双链cDNA，加入poly-A，筛选片段，富集cDNA文库。Qubit 3.0荧光计用于初步定量。对这些reads进行过滤，以获得用于基因表达和结构分析的干净高质量reads。选择显示>2倍差异(FC>2)和p<0.05的基因进行进一步分析。使用Metascape(http://metascape.org/gp/index.html)进行基因本体(GO)分析。
7、荧光素酶报告实验
使用Vazyme Biotech Co.，Ltd.(DL101-01；中国南京)的双萤光素酶报告试剂盒。将293FT细胞接种到24孔板中，当细胞密度长至20-40％时，将质粒转染到细胞中并培养48小时。接下来，丢弃细胞培养基，用PBS洗涤细胞两次，并在22℃下加入1×细胞裂解缓冲液5分钟。然后将细胞在22℃下以12000×g离心2分钟，收集上清液进行后续检测。将20微升细胞裂解物上清液添加到标准96孔板中，然后向每个孔中添加100μl萤光素酶底物。将该溶液快速混合，并立即使用GloMax Discover System(Promega，美国)检测萤火虫萤光素酶基因活性。接下来，将100μl Rinella底物工作溶液(新鲜制备)添加到反应溶液中，快速混合，然后使用GloMax Discover System(Promega，美国)立即检测Rinella萤光素酶活性。
8、统计学
使用GraphPad Prism 8进行数据分析和绘图。方差分析用于比较两组数据之间的差异。在统计学中，p＜0.05被认为具有统计学意义，分别用*、**和***表示p＜0.05、p＜0.01和p＜0.005。数据以平均值±标准差表示。所有实验都独立重复至少三次。
实施例1、鉴定TP63变异，研究TP63的14bp缺失的剪接效应
研究对象、全外显子组测序及分析：
本实施例的研究队列涉及早发性卵巢功能不全患者93人。如果患者至少有4个月的月经过少/闭经，患者年龄在40岁以下，并且如果连续两次FSH测量值>25IU/L，间隔>4周，则诊断为POI。如果POI患者表现出以下任何一种情况，则将其排除在研究之外：核型异常(X染色体异常)、自身免疫性疾病、放疗和化疗史或骨盆手术史。
基因组DNA的提取和后续的全外显子组测序及比对由诺禾致源测序公司进行。在分析数据时用于筛选的标准是错义、无义、移码或剪接位点变异以及次要等位基因频率<1％的变异。其次参考以下数据库获得等位基因频率数据：基因组聚集数据库(gnomAD，http://gnomad.broadinstitute.org/)、NHLBI外显子测序项目(ESP6500)和千人基因组项目(1000G，http://browser.1000genomes.org/index.html)。Sanger测序用于验证POI患者的TP63突变。
对以上每个93例POI患者外周血提取的DNA进行全外显子组测序(WES)。在1例POI患者的TP63基因中发现了一个14bp的杂合缺失变异c.1742_1746+9del。该患者的两次激素检测(间隔超过1年)，FSH值为137.48IU/L以及110.11IU/L。通过Sanger测序验证了患者携带的变异(图1)。这个变异覆盖了外显子和内含子的边界区域，可能会影响mRNA的剪接过程。
实施例2、TP63截短蛋白具有促进细胞凋亡的功能
本实施例构建了含有TP63基因的外显子12、内含子12、外显子13、内含子13和外显子14的WT质粒。又构建含有c.1742_1749+9del变体的突变质粒，使用重叠PCR将c.1742_1749+9del突变位点引入野生型序列中以获得突变质粒。Minigene结构示意图如图2。
随后将pcDNA3.1空载体、WT和Mut的minigene质粒转染到293FT细胞中。表达WTminigene质粒的细胞可以剪接内含子并形成正常的外显子12-13-14转录物。然而表达mutminigene质粒的细胞产生外显子12-14转录物(图3)。通过Sanger测序进一步验证了Mutminigene质粒表达细胞中跳过的外显子13(图4)，也即，14bp的缺失变异(TP63突变)导致外显子13被越过，预测产生TP63截短蛋白p.S551Rfs*6(TP63突变体)。
为验证TP63截短蛋白的功能，构建TP63野生型(TP63-WT)、TP63-mut载体。TP63野生型(TP63-WT)包含外显子12、内含子12、外显子13、内含子13和外显子14的共5508碱基对序列，该序列具有ATG(起始密码子)和TGA(终止密码子)序列；此外，使用XhoI(5′)和BamHI(3′)限制性内切酶消化位点将位于整个序列侧翼的限制性内切酶序列构建到pcDNA3.1载体中。在pcDNA3.1-3×Flag载体中还构建了编码p.S551*TP63截短蛋白的突变序列(TP63-mut)，序列如SEQ ID NO.3所示。
将相同量的TP63野生型(TP63-WT)、TP63-mut或空载体(NC)转染到293FT细胞中，发现TP63-mut在mRNA和蛋白质水平上的表达水平均低于TP63-WT(图5)。
通过Annexin V测定，我们发现15.49％的表达TP63-mut的细胞凋亡；然而，只有4.98％的表达TP63-WT的细胞凋亡(图6)，表明TP63-mut蛋白增加了细胞凋亡。Annexin V在表达TP63-mut和TP63-WT的细胞中的免疫染色也表明TP63-mut蛋白增强了细胞凋亡(图7)。
实施例3、RNA-seq分析显示CLCA2是TP63-mut蛋白的下游靶基因
为了进一步阐明TP63-mut诱导细胞凋亡的分子机制，进行RNA-seq分析。共有404个基因在TP63-mut和TP63-WT表达细胞之间差异表达，其中265个基因和139个基因分别在TP63-mut表达细胞中以较高或较低的表达水平表达(图8A-C)。265个高表达基因的基因本体(GO)分析表明，TP63途径显著富集(图8D)，表明TP63-mut蛋白比TP63-WT蛋白具有更高的基因转录活性。
在TP63-mut表达细胞中的265个高表达基因中，CLCA2是最显著的基因。qPCR分析显示，与表达TP63-WT的细胞相比，CLCA2在表达TP63-mut的细胞中以非常高的水平表达(图9A)。蛋白质印迹证实CLCA2蛋白在表达TP63-mut的细胞中表达更高(图9B)。
pGL3-CLCA2启动子质粒的构建：通过生物信息学分析和网站信息https://www.genecards.org/和https://genome.ucsc.edu/，CLCA2启动子的500bp序列被确定为TP63基因调控的区域，并设计引物(F-atttGCTAGCtttaACTGATGGAGGAGGTTATGAA；R-ggccCTCGAGcgcgAGGTTTTAGAACACAAGATGAAGG)。使用PCR扩增含有CLCA2启动子区的序列，并使用NheI(5′)和Xho1(3′)限制性内切酶消化位点将整个序列侧翼的限制性内切酶序列构建到报告基因载体(pGL3-Basic质粒)中。
为了进一步阐明TP63-mut蛋白是否可以直接转录激活CLCA2基因表达，本实施例进行了荧光素酶测定，并且与表达TP63-WT的细胞相比，在表达TP63-mut的细胞中发现了直接且更高的荧光素酶活性(图9C)。因此CLCA2是TP63-mut蛋白的下游靶标。
实施例4、敲低CLCA2可降低TP63-mut蛋白诱导的细胞凋亡
通过在293FT细胞(2.5×105个细胞/孔)中转染特异性siRNA 48小时进行CLCA2敲低。根据制造商的实验步骤使用jetPrime(101000046，Polyplus，法国)转染siRNA至细胞中。将非特异性的siRNA作为实验的对照组。转染后将细胞培养48小时用于后续的分析和测定。siCLCA2-1序列为UGACAAACCUUUCUACAUA(如SEQ ID NO:1所示)，siCLCA2-2序列为GGAAUUAUCACGUCUUACA(如SEQ ID NO:2所示)。
本实施例为了确定CLCA2是否参与TP63-mut蛋白诱导的细胞凋亡，使用特异性小干扰RNA(siRNA)来敲低CLCA2的表达。qPCR显示，在TP63-mut过表达的情况下，与siScramble组相比，siCLCA2-1和siCLCA2-2都可以降低约80％的CLCA2表达(图10A)。蛋白质印迹分析显示，siCLCA2-1和siCLCA2-2都有效地降低了CLCA2蛋白的表达(图10B)。siCLCA2-2显著降低了TP63-mut蛋白表达诱导的细胞凋亡(图10C、10D)，表明CLCA2可能是TP63-mut下游靶基因，其介导由TP63-mut蛋白表达引起的细胞凋亡。
实施例5、ATM抑制剂可降低CLCA2的表达并抑制TP63-mut蛋白表达诱导的细胞凋
亡
ATM-TP63通路被证明在消除由双链DNA断裂(DSBs)诱导的小鼠卵母细胞中发挥关键作用。据报道，ATM可以磷酸化TP63，使用ATM抑制剂(ATMi)以剂量依赖的方式抑制TP63的四聚化。因此，本实施例想确定ATMi是否减少了TP63-mut蛋白诱导的细胞凋亡。在过表达TP63-mut蛋白的情况下，将ATMi添加到细胞培养基中显著降低了CLCA2的表达(图11B)。萤光素酶测定还表明ATMi减弱了TP63-mut蛋白与CLCA2启动子的结合(图11A)。Annexin V测定表明ATMi处理显著降低了由TP63-mut蛋白过表达诱导的细胞凋亡(图11C-D)。
因此，本实施例认为TP63截短蛋白可以诱导由CLCA2的转录激活介导的细胞凋亡(图11A)，并且使用siCLCA2或ATMi处理沉默CLCA2可以显著降低CLCA2表达并抑制由TP63-mut蛋白表达诱导的细胞凋亡(图12B)。
ATM抑制剂(KU55933)从MCE公司购买(HY-12016；MCE,Monmouth Junction,NJ,USA)。
Claims (25)
1.检测CLCA2表达量的试剂在制备诊断早发性卵巢功能不全产品中的应用，所述早发性卵巢功能不全是TP63突变型的早发性卵巢功能不全，所述TP63突变型的早发性卵巢功能不全患者具有TP63突变或表达TP63突变体，所述TP63突变是c. 1742_1746+9del，所述TP63突变体的序列如SEQ ID NO.3所示。
2.如权利要求1所述应用，所述CLCA2表达量为mRNA表达量和/或蛋白表达量。
3.如权利要求2所述应用，检测所述mRNA表达量的试剂为以下方法中所使用的试剂：基于PCR的检测方法、Southern杂交方法、Northern杂交方法、点杂交方法、荧光原位杂交方法、DNA微阵列方法、ASO法或高通量测序平台方法。
4.如权利要求2所述应用，检测所述蛋白表达量的试剂为以下方法中所使用的试剂：ELISA检测、Elispot检测、Western印迹或表面等离子共振法。
5.如权利要求1所述应用，所述诊断是通过收集受试者样本进行检测而进行的，所述样本为外周血、组织、血清、血浆、尿液、唾液、精液、乳汁、脑脊髓液、泪液、痰、粘液、胞液、腹水、胸膜积液、羊水、膀胱冲洗液或支气管肺泡灌洗液。
6.CLCA2抑制剂在制备治疗早发性卵巢功能不全的产品中的应用，所述早发性卵巢功能不全是TP63突变型的早发性卵巢功能不全，所述TP63突变型的早发性卵巢功能不全患者具有TP63突变或表达TP63突变体，所述TP63突变是c. 1742_1746+9del，所述TP63突变体的序列如SEQ ID NO.3所示。
7.如权利要求6所述应用，所述CLCA2抑制剂为结合CLCA2的抗体、反义寡核苷酸、低分子量分子、siRNA、适配体或小分子化合物。
8.如权利要求6所述应用，所述CLCA2抑制剂是靶向CLCA2的siRNA。
9.如权利要求8所述应用，所述靶向CLCA2的siRNA序列如SEQ ID NO.1或SEQ ID NO.2所示。
10.如权利要求6所述应用，所述产品是药物或药物组合物，所述药物或药物组合物还包含任选一种或多种药学上可接受的载体或稀释剂。
11.如权利要求10所述应用，所述载体为固体、凝胶或液体。
12.如权利要求11所述应用，所述固体载体为乳糖、白土、蔗糖、滑石、明胶、琼脂、果胶、阿拉伯胶、硬脂酸镁、硬脂酸或可降解聚合物。
13.如权利要求11所述应用，所述液体载体为磷酸盐缓冲盐溶液、糖浆、油、水、乳剂、润湿剂或无菌溶液。
14.如权利要求10所述应用，所述稀释剂为蒸馏水、生理盐水、林格氏溶液、葡萄糖溶液、PBS溶液或汉克氏溶液。
15.如权利要求10所述应用，所述药物或药物组合物的剂型为片剂、丸剂、粉剂、颗粒剂、胶囊剂、锭剂、糖浆剂、乳剂、混悬剂、控制释放制剂、气雾剂、膜剂、注射剂、静脉滴注剂、软膏剂、洗剂、栓剂、鼻制剂、肺制剂或眼睛滴剂。
16.一种非治疗目的抑制细胞凋亡的方法，所述方法包括将CLCA2抑制剂与目标细胞接触，所述目标细胞是具有TP63突变的细胞或者是表达TP63突变体的细胞，所述突变是c.1742_1746+9del，所述TP63突变体如SEQ ID NO.3所示。
17.如权利要求16所述的方法，所述CLCA2抑制剂为结合CLCA2的抗体或适配体。
18.如权利要求16所述方法，所述CLCA2抑制剂为结合CLCA2的反义寡核苷酸或siRNA。
19.如权利要求16所述的方法，所述CLCA2抑制剂是靶向CLCA2的siRNA。
20.如权利要求19所述的方法，所述靶向CLCA2的siRNA序列如SEQ ID NO.1或SEQ IDNO.2所示。
21.如权利要求16所述的方法，所述接触是指通过转染的方式使CLCA2抑制剂进入细胞。
22.TP63突变体的检测试剂在制备诊断早发性卵巢功能不全产品的应用，所述TP63突变是c. 1742_1746+9del，所述TP63突变体的序列如SEQ ID NO.3所示。
23.如权利要求22所述应用，所述产品为试剂盒、芯片或试纸。
24.如权利要求22所述应用，所述检测试剂为引物。
25.如权利要求22所述应用，所述诊断是通过收集受试者样本进行检测而进行的，所述样本为外周血、组织、血清、血浆、尿液、唾液、精液、乳汁、脑脊髓液、泪液、痰、粘液、胞液、腹水、胸膜积液、羊水、膀胱冲洗液或支气管肺泡灌洗液。
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| CN111979315A (zh) * | 2019-05-24 | 2020-11-24 | 上海市肿瘤研究所 | 环状tp63作为肺鳞癌诊断或治疗靶点的应用 |
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| CN111549116A (zh) * | 2020-05-18 | 2020-08-18 | 王雪峰 | 女性早发性卵巢功能不全易感基因检测模型及检测试剂盒 |
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